Polynucleotide Based Movement, Kits and Methods Related Thereto

ABSTRACT

This disclosure relates DNA based movement of objects. In certain embodiments, particles, pairs of particles, or a rods are conjugated with single stranded DNA that hybridizes to a single stranded RNA that is conjugated to a substrate. When the DNA particle, pair of particles, or rod interacts with the surface RNA in the presence of an endonuclease, such as RNase H and the DNA hybridizes to the RNA, then the particle, pair of particle, or rod moves along the surface. The complementarity of the DNA and RNA affect the velocity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Number62/245,618 filed Oct. 23, 2015. The entirety of this application ishereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under R01-GM097399awarded by NIH. The government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 15080US ST25.txt. The text file is 2 KB, wascreated on Oct. 24, 2016, and is being submitted electronically viaEFS-Web.

BACKGROUND

Converting chemical energy into controlled motion is useful inapplications such as sensors, drug delivery platforms, and computing.Bath et al. report a linear motor built from DNA and a restrictionenzyme, which moves a DNA cargo in discrete steps along a DNA track.Angew Chem Int Edit 44, 4358-4361 (2005). DNA-based machines that walkalong a track have shown promise in recapitulating the properties ofbiological motor proteins. See Yin et al. Programming biomolecularself-assembly pathways. Nature, 2008, 451, 318-U314; Cha et al., NatNanotechnol, 2014, 9, 39-43; Omabegho et al., Science, 2009, 324, 67-71;Gu et al., Nature, 2010, 465, 202-205; and Lund et al., Nature, 2010,465, 206-210. However, the maximum distance traveled by the mostDNA-based motors is 1 μm. The velocity of these walkers is also limiteddue to a fundamental trade-off between motor endurance and speed. Thus,there is a need to identify improved architectures.

SUMMARY

This disclosure relates DNA based movement of objects. In certainembodiments, particles, pairs of particles, or a rods are conjugatedwith single stranded DNA that hybridizes to a single stranded RNA thatis conjugated to a substrate. When the DNA particle, pair of particles,or rod interacts with the surface RNA in the presence of anendonuclease, such as RNase H and the DNA hybridizes to the RNA, thenthe particle, pair of particle, or rod moves along the surface. Thecomplementarity of the DNA and RNA affect the velocity. In certainembodiments, this disclosure contemplates amplifying a sample nucleicacid into single stranded DNA and conjugating it to the particle, pairof particles, or a rod. Exposing the particle to complementary surfaceRNA and measuring the velocity which implicates the nucleic acidsequence in the sample.

In certain embodiments, this disclosure relates to devices comprising, aparticle, pair of particles or rod comprising a coating of singlestranded DNA; a substrate comprising a coating of single stranded RNA;and an endoribonuclease such as RNase H, wherein the single stranded DNAhybridizes to the RNA on the substrate and the particle, pair ofparticles or rod is configured on the substrate such that the particle,pair of particles, or rod moves upon mixing the endoribonuclease withthe DNA hybridized to the RNA.

In certain embodiment, the substrate comprises channels configure to beslightly greater than the diameter of the particle such that theparticle, length of a pair of particles, or length of the rod. Incertain embodiments, the channels are separated by a barrier ofpolyethylene glycol. In certain embodiment, the channels are configuredsuch that the object is capable of moving in the channel but isrestricted from isolating itself from RNA on the substrate surface.

In certain embodiments, the DNA is between 5 and 500, or 5 and 50, or 5and 25, or 10 and 50, or 10 and 25 nucleotides in length. In certainembodiments, the particle comprises silica or a semiconductor material,metal or oxide having a polymer coating.

In certain embodiments, the DNA oligonucleotide is 3′ or 5′ conjugatedto the surface of the particle. In certain embodiments, the particle,pair of particles, or rod has a diameter or length of 0.001 micrometersto 1 centimeters, or 0.001 micrometers to 0.1 centimeters, or 0.001micrometers to 0.01 centimeters, or 0.001 micrometers to 1 micrometer.In certain embodiments, the DNA or the RNA encodes a polynucleotidesequence associates with a polymorphism, SNP, or mutation associatedwith a genetic disorder. In certain embodiments, the DNA or RNA canencode aptamer or split aptamer sequences associated with binding toaptamer ligand. In certain embodiments, DNA or RNA sequences encodes acatalytic oligonucleotide associated with specific metal cofactors. Incertain embodiments, the substrate is a metal surface, glass, polymer,or microscope slide. In certain embodiments, the RNA is conjugated to afluorescent molecule. In certain embodiments, movement of the particle,pair of particles, or rods are measured for velocity, e.g., randommovement or in a single direction.

In certain embodiments, the disclosure relates to methods for moving aparticle, pair or particles, or rod comprising DNA, comprising:providing a device comprising, a particle, pair of particles, or rodcomprising a coating of single stranded DNA; a substrate comprising acoating of single stranded RNA; and an endoribonuclease such as RNase H,wherein the single stranded DNA hybridizes to the RNA on the substrateand the particle, pair of particles, or rod is configured on thesubstrate such that the particle, pair of particles, or rod moves uponmixing the endoribonuclease with the DNA hybridized to the RNA; placingthe single stranded DNA coated particle, pair or particles, or rod onthe surface of the single stranded RNA coated substrate in the presenceof the endonuclease under conditions such that the particle, pair ofparticles, or rod moves on the surface of the substrate.

In certain embodiments, DNA or RNA is a sequence obtained from a sample.In certain embodiments, the speed of the movement of the particle, pairof particles, or rod is correlated to the sequence of the DNA or RNA. Incertain embodiments, a maximum speed is associated with completecomplementarity. In certain embodiments, a speed of less than themaximum speed is associated with incomplete complementarity.

In certain embodiments, the disclosure relates to kits comprising: a) apair or primers wherein the primers are configured for amplification ofa target DNA sequence, b) a substrate that contains single stranded DNAbound to the surface, and c) an oligonucleotide conjugated to afluorescent marker wherein the oligonucleotide has a first segment thatis complementary to the DNA bonded to the surface of the substrate and asecond segment that is RNA and complementary to the target DNA sequencethat is to be obtained from a sample by amplification from the pair ofprimers.

In certain embodiments, the kit further comprises RNase H.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A illustrates an approach for generating RNA-fueled, enzymecatalyzed autonomous DNA motors. DNA-modified particles were hybridizedto an RNA monolayer presenting a complementary strand. Particles wereimmobile until RNase H was added, which selectively hydrolyses RNAduplexed to DNA. Schematic representation showing the hybridizedoligonucleotide sequences at the particle-substrate junction. Note thatthere are hundreds of duplexes within this junction.

FIG. 1B shows a figure with oligonucleotide sequences conjugated to dyesand other molecules for support on solid surfaces.

FIG. 1C illustrates the chemical structures of the molecules conjugatedto the oligonucleotides.

FIG. 2A shows bar graph with data indicating the DNA anchor strandincubation concentration alters the Cy3-RNA fluorescence intensity whichis directly proportional to the RNA surface density. The maximum RNAsurface density was achieved when the DNA anchor strand incubationconcentration was equal to or greater than 1 μM. Error bars representthe standard deviation in the average fluorescence intensity from atleast 5 regions across each channel.

FIG. 2B shows a plot indicating the RNA surface density as a function ofRNA concentration during Cy3-RNA hybridization with surface immobilizedDNA anchor strand. The RNA density was maximized when RNA was hybridizedat a concentration of at least 100 nM.

FIG. 2C shows a representative BF image and trajectory (line) from atime-lapse video tracking a single microparticle 30 min following RNaseH addition. The same region was then imaged in the Cy3 fluorescencechannel, revealing the location of depleted Cy3 signal.

FIG. 2D shows a line scan plot of dashed white line from FIG. 2C showingthe depletion track from the widefield fluorescence image.

FIG. 2E shows a histogram analysis of FWHM of the depletion path widthacquired using structured illumination microscopy.

FIG. 2F shows data on the MSD versus log (time) analysis from individualparticle trajectories (n =43), which is shown with black circles. Theline indicates the average slope derived from all the individualparticle trajectories.

FIG. 3A schematically illustrates the strategy used to test whetherparticles roll during translocation by blocking the free DNA of theparticle by hybridizing with a blocking DNA strand.

FIG. 3B shows a representative BF image and trajectory taken from atime-lapse video tracking a single particle that had been blocked withDNA and treated with RNase H. The same region was imaged using the Cy3fluorescence channel, indicating the lack of a RNA hydrolysis track.Note that a small transient spot with lower fluorescence intensity (seecenter of fluorescence image) is typically observed under particles andis not due to RNA hydrolysis.

FIG. 3C shows MSD versus log(time) plot of particle diffusion for theblocked particles. The black circles represent individual data pointsfrom n =32 particles, while the line indicates the average of theseplots.

FIG. 3D shows a histogram analysis of particle velocity for each 5sinterval as a function of [KCl]; 38 mM (n=43 particles (15,480occurrences)) and 150 mM (n=52 particles (18,720 occurrences)).

FIG. 3E shows a histogram analysis of particle velocity for each 5 sinterval as a function of pH; 8.0 (n=43 particles (15,480 occurrences)),7.5 (n=50 particles (18,000 occurrences)), and 7.0 (n=26 particles(9,360 occurrences)). Inset compares RNase H kcat and average particlevelocity as a function of pH.

FIG. 3F shows a representative BF image and trajectory taken from atime-lapse video tracking a single particle for 30 min where the sectionindicates when the particle becomes entrapped.

FIG. 3G shows a representative velocity histogram of an individualparticle when the particle is entrapped or not entrapped. Entrapmentleads to significant decrease in particle velocity.

FIG. 3H shows a plot of the particle velocity dependence on RNase Hconcentration. Note that in the absence of RNase H, the particles do notmove.

FIG. 4A shows directional motor translocation by time-lapse images of aparticle moving along a 3 μm wide track following the addition of RNaseH. A strategy was used to generate RNA micro-tracks by usingmicrocontact printing. SH-PEG barriers were directly printed onto thegold film, which was backfilled with RNA.

FIG. 4B shows a line scan analysis of the region highlighted in thefluorescence channel with a dotted white line showing the trackdimensions and the effectiveness of the PEG barriers.

FIG. 4C shows representative BF and fluorescence images taken from atime-lapse movie that tracked a dimerized particle following RNase Haddition. The BF-analyzed trajectory as well as the two parallelfluorescence depletion tracks showed near linear particle motion.

FIG. 4D shows MSD versus log(time) analysis of the dimerized particlemotion. Slope of this plot shows an average power log dependence of1.82±0.13, confirming that the particle dimer traveled in a ballistic,linear fashion.

FIG. 5 illustrates an example of single nucleotide polymorphism (SNP)detection using bright field images. The images show particles and theirnet displacement for perfect match and SNP sequences after RNase Haddition. The sequences are illustrated below each.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

An oligonucleotide is a plurality of joined nucleotides joined by nativephosphodiester bonds, between about 6 and about 300 nucleotides inlength. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA.

As used herein, biological samples include all clinical samples usefulfor detection of disease in subjects, including, but not limited to,cells, tissues, and bodily fluids, such as: blood; derivatives andfractions of blood, such as serum; extracted galls; biopsied orsurgically removed tissue, including tissues that are, for example,unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears;milk; skin scrapes; surface washings; urine; sputum; cerebrospinalfluid; prostate fluid; pus; or bone marrow aspirates. In a particularexample, a sample includes blood obtained from a human subject, such aswhole blood or serum. In another particular example, a sample includesbuccal cells, for example collected using a swab or by an oral rinse.

Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

A mutation refers to a change of a nucleic acid sequence as a source ofgenetic variation. For example, mutations can occur within a gene orchromosome, including specific changes in non-coding regions of achromosome, for instance changes in or near regulatory regions of genes.Types of mutations include, but are not limited to, base substitutionpoint mutations (which are either transitions or transversions),deletions, and insertions. Missense mutations are those that introduce adifferent amino acid into the sequence of the encoded protein; nonsensemutations are those that introduce a new stop codon; and silentmutations are those that introduce the same amino acid often with a basechange in the third position of the codon. In the case of insertions ordeletions, mutations can be in-frame (not changing the frame of theoverall sequence) or frame shift mutations, which may result in themisreading of a large number of codons (and often leads to abnormaltermination of the encoded product due to the presence of a stop codonin the alternative frame).

Polymorphism refers to a variation in a gene sequence. The polymorphismscan be those variations (DNA sequence differences) which are generallyfound between individuals or different ethnic groups and geographiclocations which, while having a different sequence, produce functionallyequivalent gene products. Typically, the term can also refer to variantsin the sequence which can lead to gene products that are notfunctionally equivalent. Polymorphisms also encompass variations whichcan be classified as alleles and/or mutations which can produce geneproducts which may have an altered function. Polymorphisms alsoencompass variations which can be classified as alleles and/or mutationswhich either produce no gene product or an inactive gene product or anactive gene product produced at an abnormal rate or in an inappropriatetissue or in response to an inappropriate stimulus. Alleles are thealternate forms that occur at the polymorphism.

A “single nucleotide polymorphism (SNP)” is a single base (nucleotide)polymorphism in a DNA sequence among individuals in a population.Typically in the literature, a single nucleotide polymorphism (SNP) mayfall within coding sequences of genes, non-coding regions of genes, orin the intergenic regions between genes. SNPs within a coding sequencewill not necessarily change the amino acid sequence of the protein thatis produced, due to degeneracy of the genetic code. A SNP in which bothforms lead to the same polypeptide sequence is termed “synonymous”(sometimes called a silent mutation)—if a different polypeptide sequenceis produced they are “nonsynonymous”. A nonsynonymous change may eitherbe missense or “nonsense”, where a missense change results in adifferent amino acid, while a nonsense change results in a prematurestop codon.

Hybridization refers to the ability of complementary single-stranded DNAor RNA to form a duplex molecule (also referred to as a hybridizationcomplex). Nucleic acid hybridization techniques can be used to formhybridization complexes between a probe or primer and a nucleic acid.Hybridizable and hybridizes are terms which indicate a sufficient degreeof complementarity such that stable and specific binding occurs betweenan oligonucleotide and its DNA or RNA target. An oligonucleotide neednot be 100% complementary to its target DNA or RNA sequence to bespecifically hybridizable. An oligonucleotide is specificallyhybridizable when there is a sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions in which specific binding is desired.Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.

Primers are short nucleic acids, preferably DNA oligonucleotides 15nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand, and then extendedalong the target DNA strand by a DNA polymerase enzyme. Primer pairs canbe used for amplification of a nucleic acid sequence, e.g., by thepolymerase chain reaction (PCR) or other nucleic-acid amplificationmethods known in the art. The primers disclosed herein can hybridize tonucleic acid molecules under low stringency, high stringency, and veryhigh stringency conditions.

As used herein, Ribonuclease H (RNase H) is a family ofnon-sequence-specific endonucleases that catalyze the cleavage of RNAvia a hydrolytic mechanism. Ribonuclease activity for RNase H cleavesthe 3′-0-P bond of RNA in a DNA/RNA duplex substrate to produce 3′-hydroxyl and 5′ -phosphate terminated products and the RNase Hspecifically degrades only the RNA in RNA:DNA complex.

As used herein, conjugated refers to either covalently attaching two orobjects together, or creating hydrogen bonding interactions, such ashybridization of two nucleic acids, such that the two objects do notsubstantially dissociate in a solution of water at room temperature andneutral pH.

In certain embodiments, this disclosure relates to devices comprising, aparticle, pair of particles, or rod comprising a coating of singlestranded DNA; a substrate comprising a coating of single stranded RNA;and an endoribonuclease such as RNase H, wherein the single stranded DNAhybridizes to the RNA on the substrate and the particle, pair ofparticles, or rod is configured on the substrate such that the particle,pair of particles or rod, moves upon mixing the endoribonuclease withthe DNA hybridized to the RNA.

In certain embodiments, the particle is a conglomerate of matter that ispreferably spherical in shape. However, it is contemplated that theparticle may not be perfectly spherical, e.g. oval or havingimperfections. The diameter of a particle refers to the average size ofthe diameter. Typically the size of the particle is such that thelocation of the particle can be readily identified by visual or otherspectroscopic means on a substrate. The particle may be made of a corematerial that has covalent bonds, e.g., polymers or resins, or fromsemiconductor materials, e.g., CdSe, CdS, CdTe quantum dots, metals oroxides thereof, e.g., iron oxide particles.

In certain embodiments, a “rod” refers two or more particles that arejoined together to form a length that is two or more times the smallestdiameter of one of the particles. The rod may be straight or have aslight bend, for example when three or more particles are joined but donot exist in an absolute straight line. In certain embodiments, a rodmay be nanotube or other structure that can be conjugated with DNA.

In the case of quantum dot, metal particle or rod, the outer surface mayhave a polymer coating that is chemically crosslinked to prevent thepolymers from separating from the particle or rod.

In certain embodiments, the “coating of single stranded DNA” refers tothe conjugation of a nucleic acid to the outer surface of a particle,pair of particles, or rod wherein at least a portion of the DNA sequenceis single stranded. The DNA may be hybridizing to a complementary strandthat is used to conjugate the DNA to the particle providing a portion ofthe DNA that is double stranded.

In certain embodiments, a “substrate” refers to a surface that isstationary with respect to the RNA conjugated thereto. Conjugation ofsingle stranded RNA to the substrate may be by hybridization or bycovalently linking the RNA. The surface may be planar or curved so longas the surface area is sufficiently large in relation to any particlesor rods placed thereon such that movement of the particles or rods fromdifferent locations on the surface can be detected.

Methods of Use

As illustrated in FIG. 1, a sample of DNA is isolated from a subject andthe 5′ end of the DNA has been modified with an alkynyl group to allowcoupling of the DNA to the surface of a particle comprising azides toform 1,2,3-triazoles. Providing a particle comprising a single strandedDNA. The substrate comprises a gold surface which is bound through aterminal thiol to a DNA that is hybridized to an RNA. Because the RNA islonger sequence than the substrate bound DNA, the substrate provides aportion of single stranded RNA. The single stranded RNA is capable ofhybridizing to the single stranded DNA on the surface particle.Therefore, when the particle is places on the surface of the substratethe single stranded DNA and single stranded RNA bind. Further in thepresence of RNase H, the RNA is degraded freeing the DNA on the particlefor subsequent movement of the particle through new interactions of DNAand RNA on the surface.

In certain embodiments, the disclosure relates to methods for moving aparticle, pair of particles, or rod comprising DNA, comprising:providing a device comprising, a particle or rod comprising a coating ofsingle stranded DNA; a substrate comprising a coating of single strandedRNA; and an endoribonuclease such as RNase H, wherein the singlestranded DNA hybridizes to the RNA on the substrate and the particle,pair of particles, or rod is configured on the substrate such that theparticle, pair of particles, or rod moves upon mixing theendoribonuclease with the DNA hybridized to the RNA; placing the singlestranded DNA coated particle on the surface of the single stranded RNAcoated substrate in the presence of the endonuclease under conditionssuch that the particle, pair of particles, or rod moves on the surfaceof the substrate.

In certain embodiments, DNA or RNA is a sequence obtained from a sample.In certain embodiments, the speed of the movement of the particle iscorrelated to the sequence of the DNA or RNA. In certain embodiments, amaximum speed is associated with complete complementarity. In certainembodiments, a speed of less than the maximum speed is associated withincomplete complementarity. In certain embodiments, a pair of primersare used to obtain a predetermined DNA sequence from a sample of thesubject. The primers may target RNA, e.g., mRNA, or DNA that is in thesample.

In certain embodiments, the disclosure contemplates a kit comprising: apair or primers, a substrate that contains a DNA bound to the surface,and an oligonucleotide conjugated to a fluorescent marker wherein theoligonucleotide has a first segment that is complementary to the DNAbonded to the surface of the substrate and a second segment that iscomplementary to a DNA sequence that is to be obtained from a sample byapplication from the pair of primers. The DNA sequence that is to beobtained from the sample is amplified from a pair of primers. Thesequences of the primers may be removed prior to placing the singlestranded DNA on the particle through the use of sequence specificrestrictions enzymes built into the primers.

Other configurations are contemplated such as the particle, pair ofparticles, or rod having a capture DNA already attached to the particle.In certain embodiments, the disclosure relates to kits comprising: aparticle, pair or particles, or rod comprising a capture DNA; asubstrate comprising a coating of single stranded RNA; and a pair ofprimers configures to amplify a nucleic acid that is complementary tothe single stranded RNA and an endoribonuclease. In certain embodiments,the pair of primers is a first primer and a second primer, wherein thefirst primer is a sequence that has a sequence of five or morenucleotides that are identical or complementary to the single strandedDNA conjugated to the particle, pair of particles, or rod and the secondprimer has a sequence of five or more nucleotides that are identical orcomplementary to a sequence of the single stranded RNA.

In certain embodiments, the disclosure relates to kits comprising: aparticle, pair or particles, or rod comprising a group reactive the 3′of 5′ end of single stranded DNA; a substrate comprising a coating ofsingle stranded RNA; and a pair of primers configures to amplify anucleic acid that is complementary to the single stranded RNA and anendoribonuclease.

Design and Synthesis of Spherical RNase H Powered Motors

In certain embodiments, an embodiment of this disclosure is a motorwhich consists of a DNA-coated spherical particle (5 μm or 0.5 μmdiameter particles) that hybridizes to a surface modified withcomplementary RNA. The particle moves upon addition of RNase H, whichselectively hydrolyses hybridized RNA but not single stranded RNA. Sincethe driving force for movement is derived from the free energy ofbinding new single stranded RNA that biases Brownian motion away fromconsumed substrate (FIG. 1A), this type of motion is often described asa “burnt-bridge Brownian ratchet”. Note that molecular walkers alsoemploy a burnt bridge mechanism, where oligonucleotide hybridization isfollowed by DNAzyme/endonuclease hydrolysis of the fuel strand. The maindifference between the molecular walkers and our system is the massivemultivalency of the DNA coated particles—molecular walkers typicallyemploy 2-6 anchor points while our particle-based motor employsthousands of anchoring strands. This equates to 100-1000 fold greatercontact area or 10-100 fold greater contact diameter with the surfacewhen compared to molecular spiders. Hence, the micron sized length scaleof the particle significantly increases the number of contacts with thesurface, which should lead to collective and emergent properties notexpected for DNA-based walkers.

Highly multivalent motors display greater processivity, thus addressinga major limitation of DNA walkers. The spherical particle templateallows for the potential to roll, which is a fundamentally differentmode for translocation of DNA based machines.

An RNA-monolayer was generated on a substrate by immobilizing a DNAanchor strand to a thin gold film and then hybridizing a fluorescentlylabeled RNA-DNA chimera strand to the surface. A Cy3 fluorophore at the3′ RNA terminus was used to optimize RNA density and to detect RNAhydrolysis using fluorescence microscopy (FIG. 1B). Using the optimizedconditions, a maximum RNA density of 50,000 molecules/μm² was achieved,equivalent to an average molecular footprint of 20±6 nm² per RNA strand.This RNA density was maintained for at least 4 hrs in 1× PBS and 10 μMDTT, a thiol reducing agent necessary for maintaining RNase H activity.In the absence of DTT, surfaces were stable for weeks in 1× PBS.

Given that particle motion is intimately connected with RNase Hefficiency and enzyme rates vary when substrates are immobilized,hydrolysis kinetics were measured for a DNA-RNA duplex monolayer.Initially, when measuring the hydrolysis of surface immobilized RNA,RNase H was completely inhibited. Since RNase H contains multiplecysteine residues, it was suspected that enzyme inhibition was due toirreversible binding of the enzyme to the Au surface. To preventnonspecific binding, the Au surface was passivated withSH(CH2)₁₁(OCH2CH2)₆OCH₃ (SH-PEG) in order to reduce nonspecificinteractions with surface. To test the assumption that RNase Hinhibition was due to Au film binding, the DNA monolayer surface wasbackfilled with SH-PEG under a range of conditions, where the SH-PEGconcentration and the passivation time was varied. It was determinedthat complete surface passivation occurred after 4 hrs of incubationwith a 100 μM SH-PEG solution. This was inferred by observing asaturation in the loss of fluorescence of FAM labeled DNA anchor strand.Next, RNase H hydrolysis of surface immobilized RNA duplexed with DNAwas investigated under the various passivation conditions by measuringthe loss in fluorescence of Cy3 labeled RNA throughout the channel overtime. When the channel was SH-PEG passivated for shorter durations (2hrs), the fluorescence intensity varied significantly across the lengthof the well; regions near the port where RNase H was added had thelowest intensities, while regions furthest away from this site showedminimal substrate hydrolysis. In contrast, channels that were blockedfor 6 hrs showed homogeneous fluorescence intensities indicating uniformRNase H activity levels.

DNA-functionalized particles with a density of ˜91,000 molecules/μm′(footprint of 11±3 nm 2 per molecule) were synthesized and hybridized toa substrate presenting the complementary RNA strand. The DNA densitymatched that of the RNA density on the planar substrate to ensure a highdegree of polyvalency (˜104 contacts/μm), therefore minimizing motordetachment from the substrate and maximizing run processivity. Particlesremained immobile until RNase H was added, which led to rapidtranslocation of particles across the substrate. This was quantitativelytracked by finding the centroid of the particles in time-lapsebrightfield (BF) microscopy at 5 sec intervals (FIG. 2C). TheBF-generated tracks matched the widefield fluorescence depletion tracks(FWHM of 720±110 nm), confirming that the particle motion was associatedwith continuous RNA hydrolysis (FIG. 2C and D). The line scan analysisof the fluorescence depletion indicates ˜50% of the RNA underneath theparticle is hydrolyzed (FIG. 2D). Structured illumination microscopy(SIM), a super resolution microscopy technique with ˜110 nm resolutionrevealed a more accurate footprint of the particle substrate junctioncorresponding to an average track width of 380±50 nm (n=55 tracks). Thisfootprint indicates a maximum of ˜5,500 DNA-RNA surface contacts at themotor-substrate junction (FIG. 2E). Substrates comprised of DNA did notlead to any translocation upon addition of RNase H, confirming thatparticle motion is specific to RNA hydrolysis at the particle-substratejunction.

Unrestricted Particle Motion

Particle motion could occur through three plausible mechanisms: a)walking/sliding, b) hopping, and c) rolling. The hopping mechanism wasimmediately ruled out upon examination of the continuous fluorescencedepletion tracks (FIG. 2C). To differentiate between the two remainingmechanisms, particles were hybridized to an RNA substrate, and theunbound DNA on the particle was blocked by hybridization with acomplementary DNA strand (FIG. 3A). If motion primarily occurs through awalking/sliding mechanism, particles would move in a processive fashionleaving behind an RNA depletion track. However, upon RNase H addition,particles diffused randomly, producing an α=0.99±0.22 and a v=1.8±0.8,and no corresponding RNA depletion tracks were observed (FIG. 3B and C).By ruling out the hopping and walking/sliding mechanisms of motion,particles primarily translocate by rolling, in a monowheel orcog-and-wheel like fashion. This is the first example of a DNA-basedautonomous rolling motor, monowheels.

The particle speed histogram contains two populations (FIG. 3D). Uponfurther analysis of individual particle velocities and accounting forstage drift, the two populations correspond to two states for eachparticle, a slow/stalled state and a fast state, as opposed to two typesof particles or contributions owing to stage drift. The slower state isdue to transient stalling of the particle, which may be attributed tofactors that include surface defects leading to non-specific particlebinding, inactive enzyme bound to the particle-substrate junction, andparticle self-entrapment. Upon detailed analysis of individual particletrajectories, stalling mostly correlated to particle entrapment (FIG.3F, G). To determine whether the enzyme concentration used, [RNaseH]=140 nM, saturated the available substrate binding sites, monowheelvelocity was monitored as a function of enzyme concentration (FIG. 3h ).At 10 fold greater enzyme concentration, only a slight increase inaverage velocity was observed, whereas 10 fold and 100 fold dilution ofRNase H led to near abolition of monowheel motion. The particle speedhistograms for decreasing RNase H concentration show a gradual decreasein velocity as opposed to a shift to the low velocity population, thusconfirming that multiple RNase H enzymes are operating in parallel.

Particles of 5 μm in diameter were used for the majority of experiments.However, note that the rolling mechanism of translocation can berecapitulated with 0.5 μm diameter particles. Similar maximum velocitiesup to 5 μm/min and average velocities of 1.8±0.4 and 1.9±0.5 μm/min wereobserved for both 0.5 and 5 μm particles, respectively, showing that thefundamental cog-and-wheel mechanism of rolling is independent of cargosize within the range tested. The less multivalent 0.5 μm particles rollfor shorter average run lengths compared to 5 μm diameter particles (˜3μm versus ˜200 μm), which continue processively moving throughout the 30min video and even continue moving for over 5 hrs. Increasing the KC1and Mg concentrations to 75 mM and 3 mM, respectively, enhances 0.5 μmparticle endurance such that the majority of particles displayprocessive motion for the entire 30 min video. This provides the 0.5 μmparticles with an average run length of greater than 25 μm.

Unidirectional Motion

To achieve unidirectional transport resembling motor protein motionalong a filament, RNA was spatially micro-patterned into 3 pm widetracks. Particles were then hybridized to the patterned RNA, and RNase Hwas added to initiate motion. Using BF time-lapse tracking and RNAfluorescence depletion, a subset of particles moved along the 3 μmsubstrate corral unidirectionaly deflecting away from the PEG-printedregions was observed (FIG. 4A). Note that many particles becameentrapped, partially because of RNA cross contamination into thePEG-passivated regions (FIG. 4B) and self-entrapment in consumedsubstrate corrals. It is likely that generating well-passivatednanoscale RNA tracks commensurate in size to the particle-substratejunction width (˜400 nm) would lead to an increase yield of lineartrajectories.

An alternate strategy to achieve linear motion is to limit lateralparticle motion by incorporating multiple monowheels on the body of asingle chassis. By happenstance, it was noticed that a 1-10% subset ofour particles were fused forming dimers, a common byproduct in silicaparticle synthesis. These particles travelled linearly for distancesthat spanned hundreds of μm's at a velocity of ˜0.6±0.5 μm/min, n=68dimer particles (FIG. 4C). A plot of MSD versus t for particle dimersshowed a power-law scaling of a=1.82±0.13, confirming that particlemotion was nearly linear (α=2). In addition, 50% of single sphericalparticles displayed a transient component of their trajectory that islinear and associated with wider tracks; linear motion was correlatedwith wider ˜1.0±0.1 μm tracks or multiple contact points. The ballistic(linear) motion observed for what appears to be wider tracks may be dueto particles possessing multiple contact points that cannot be resolvedor due to particles rolling along imperfections along the surface.Following these observations, DNA-coated microrods were synthesized.Microrods showed nearly linear motion. These are the first examples ofdirectional motion without the need of a patterned track or externalelectromagnetic field, which is only afforded due to the uniquecog-and-wheel translocation mechanism.

SNP Detection by Measuring Particle Displacement

Since monowheel motion is sensitive to k_(on), k_(off) and k_(cat),particle motion could provide a readout of molecular recognition.Particles displaying the SNP (5′mAmGTAATTAAmUmC3′) traveled ˜60% slower(0.3 μm/min) than identical particles with a perfect match(5′mAmGTAATCAAmUmC3′). This difference in velocity can be attributed toa slower rate of hydrolysis for RNase H to hydrolyze duplexes possessinga single base mismatch. Due to the μm-sized cargo and large distancestravelled, even a smartphone camera equipped with an inexpensive plasticlens could detect motion associated with this SNP by recording particledisplacement within a short time interval (t=15 min). SNP detectioncould also be achieved using unmodified DNA (FIG. 5); although maximumdiscrimination required shortening the RNase H recognition sequence.

1. A device comprising, a particle, pair of particles, or a rodcomprising a coating of single stranded DNA; a substrate comprising acoating of single stranded RNA; and RNase H, wherein the single strandedDNA hybridizes to the single stranded RNA on the substrate and theparticle, pair of particles, or rod is configured on the substrate suchthat the particle, pair of particles, or rod moves upon mixing RNase Hwith the DNA hybridized to the RNA.
 2. The device of claim 1, whereinthe DNA is between 5 and 500 nucleotides in length.
 3. The device ofclaim 1, wherein the particle comprises silica or a metal having apolymer coating.
 4. The device of claim 1, wherein the particle has adiameter of 0.001 micrometers to 1 centimeters.
 5. The device of claim1, wherein the DNA or the RNA encodes a mutation associated with agenetic disorder.
 6. The device of claim 1, wherein the particle, pairof particles, or rods are measured for velocity in a single direction.7. The device of claim 1, wherein the RNA is conjugated to a fluorescentmolecule.
 8. A method for moving a particle comprising DNA, comprising:providing a device comprising, a particle, pair of particles, or a rodcomprising a coating of single stranded DNA; a substrate comprising acoating of single stranded RNA; and RNase H, wherein the single strandedDNA hybridizes to the single stranded RNA on the substrate and theparticle, pair of particles, or rod is configured on the substrate suchthat the particle moves upon mixing RNase H with the DNA hybridized tothe RNA; placing the single stranded DNA coated particle on the surfaceof the single stranded RNA coated substrate in the presence of RNase Hunder conditions such that the particle, pair of particles, or rod moveson the surface of the substrate.
 9. The method of claim 8, wherein DNAor RNA is a sequence obtained from a sample.
 10. The method of claim 9,wherein the speed of the movement of the particle is correlated to thesequence of the DNA or RNA.
 11. A kit comprising: a) a pair or primerswherein the primers are configured for amplification of a target DNAsequence, b) a substrate that contains a DNA bound to the surface, andc) an oligonucleotide conjugated to a fluorescent marker wherein theoligonucleotide has a first segment that is complementary to the DNAbonded to the surface of the substrate and a second segment that iscomplementary to the target DNA sequence that is to be obtained from asample by amplification from the pair of primers.
 12. The kit of claim11, further comprising an RNase H.